Fluorescent molecularly imprinted polymer particles for glyphosate detection using phase transfer agents

In this work, molecular imprinting was combined with direct fluorescence detection of the pesticide Glyphosate (GPS). Firstly, the solubility of highly polar GPS in organic solvents was improved by using lipophilic tetrabutylammonium (TBA+) and tetrahexylammonium (THA+) counterions. Secondly, to achieve fluorescence detection, a fluorescent crosslinker containing urea-binding motifs was used as a probe for GPS-TBA and GPS-THA salts in chloroform, generating stable complexes through hydrogen bond formation. The GPS/fluorescent dye complexes were imprinted into 2–3 nm fluorescent molecularly imprinted polymer (MIP) shells on the surface of sub-micron silica particles using chloroform as porogen. Thus, the MIP binding behavior could be easily evaluated by fluorescence titrations in suspension to monitor the spectral changes upon addition of the GPS analytes. While MIPs prepared with GPS-TBA and GPS-THA both displayed satisfactory imprinting following titration with the corresponding analytes in chloroform, GPS-THA MIPs displayed better selectivity against competing molecules. Moreover, the THA+ counterion was found to be a more powerful phase transfer agent than TBA+ in a biphasic assay, enabling the direct fluorescence detection and quantification of GPS in water. A limit of detection of 1.45 µM and a linear range of 5–55 µM were obtained, which match well with WHO guidelines for the acceptable daily intake of GPS in water (5.32 µM).


Synthesis of silica beads
Silica particles were synthesized according to the Stöber method with modifications [1]. 65 mL of 96 % ethanol, 121 mL of Milli-Q water and 14 mL of 32 % ammonia solution were mixed at 300 rpm in a 1 L Erlenmeyer flask. 18 mL of TEOS (80 mmol) was mixed with 182 mL of 96 % ethanol and quickly added to the base solution. The mixture was stirred overnight at 300 rpm. The resulting particles were washed 3 times with 96 % ethanol by centrifugation and redispersion at 12,700× g for 10 min and then dried overnight in a vacuum.

Modification of silica particles with APTES
APTES modification of silica particles was performed as previously reported [1]. 1 g of silica particles was weighed into a 2-necked round bottomed flask equipped with a magnetic stirrer and connected to a reflux condenser. The particles were dispersed in 50 mL of anhydrous toluene and heated to 120 °C under argon. 4 mL of APTES (17.1 mmol) was added and the reaction allowed to proceed for 16 h under reflux. The particles were then washed 3 times with 35 mL of 96 % ethanol, with centrifugation at 12,700× g for 5 min and 5 min sonication between washes. The particles (APTES@SiO2) were dried overnight in a vacuum.

Modification of APTES@SiO2 particles with reversible-addition fragmentation chain transfer (RAFT) agent, CPDB
RAFT modification of the APTES@SiO2 particles was performed according to previously published protocols [1]. Briefly, 500 mg of APTES@SiO2 particles were weighed into a 20 mL vial equipped with a magnetic stirrer and placed in an ice bath. Simultaneously, 117.4 mg of CPDB (0.4 mmol), 40.3 µL of ECF (0.4 mmol) and 58.7 µL of TEA (0.4 mmol) were dissolved in 8.6 mL of anhydrous tetrahydrofuran (THF) and mixed together in an acetone/liquid nitrogen bath at -78 °C for 40 min. Afterwards, the cooled solution was added to the particles and left to react at room temperature for 24 h at 700 rpm. The particles were precipitated with 15 mL of n-hexane and centrifugation performed at 12,700× g for 5 min. The particles were washed once with 20 mL of THF, once with 20 mL of acetone and in 20 mL of THF once more, with centrifugation at 12,700× g for 5 min and 5 min sonication between washes. The particles (RAFT@SiO2) were then dried under vacuum overnight.
S-3 2 NMR spectra of fluorescent crosslinker I Figure S1. 1 H NMR spectrum of I in DMSO-d6.

Determination of binding constant of I with GPS-TBA and GPS-THA
Binding constants were determined using BindFit software (http://supramolecular.org/) [2]. The program accepts input data consisting of host (dye) and guest (GPS-TXA) concentrations as well as fluorescence intensity values at the measured wavelengths. The user provides an initial guess of the binding constants, and the software uses this value to fit the data into a model using nonlinear regression. The uncertainty of the fit gives an indication of the suitability of the model obtained, and the initial guess can be refined to obtain a fit with the least uncertainty.
To determine the association constant K a fluor of I with GPS-TBA and GPS-THA, fluorescence spectra were recorded for a dilute solution of I in chloroform (4.8 µM), and a solution of GPS-TBA or GPS-THA in this dilute dye solution was prepared (9.6 mM and 3.8 mM, respectively). Small amounts of the template-dye solution were increasingly added to the dilute solution of I until saturation was reached. The fluorescence data in both cases fit a 1:1 (host:guest) binding model. The species distribution following the fitting of titration data of I with GPS-TBA and GPS-THA is shown in Figure  S4.

Selectivity of various MIP recipes by varying reactant ratios
Various recipes were tested to optimize binding performance (Table S1). GPS-TBA and MPA-TBA were used as templates to prepare the MIP and dNIP particles respectively. After titration of the particles with GPS-TBA, the relative fluorescence change, was calculated for each fluorescence spectrum of the MIP and dNIP (where F x is the fluorescence intensity at 491 nm for each spectrum after template addition, while F 0 is the fluorescence intensity at 491 nm before addition of template). The optical imprinting factor (I.F.) was then calculated for each MIP and dNIP combination, by determining the ratio of fluorescence changes of MIP and dNIP at the point of saturation. The molar ratio of template/I/MAAm/EGDMA that yielded the best results was 1:1:30:150.

Determination of amount of I incorporated into MIP and dNIP particles
Following MIP synthesis, a 15-20 nm shift in the wavelength of maximum absorption of I in MIP and dNIP particles was observed compared to I in dilute solution. Consequently, the molar absorption coefficient of fluorescent crosslinker I at a specified wavelength in chloroform could not be used to accurately determine the concentration of I in the MIP and dNIP particles. Instead, the area under the longest wavelength band of the absorption spectrum of a known S-11 concentration of I in chloroform was used as reference for the determination. Absorption spectra of three stock solutions of I in chloroform (7.39 µM) were recorded with two replicates in a quartz cuvette with 1 cm optical path length ( Figure S13). Absorption spectra of known particle concentrations of the MIP and dNIP particles were recorded in chloroform in 2 replicates in a quartz cuvette with 1 cm optical path length. For MIPTBA@SiO2 and NIPTBA@SiO2, the absorption data at the end point of the titration was used, since prior to titration a proportion of I − was present, which was converted to I after titration.

Determination of LOB and LOD of MIPTBA@SiO2 and MIPTHA@SiO2
The parameters Limit of Blank (LOB) and Limit of Detection (LOD) describe the smallest concentration of a sample that can be reliably measured by an analytical procedure [3]. LOB is defined as the highest putative analyte concentration expected to be found when replicates of a blank sample in absence of the analyte are measured. LOD is defined as the lowest analyte concentration likely to be reliably distinguished from the LOB and at which detection is feasible.
The absolute values of the fluorescence emission data at 491 nm following titration with increasing amounts of corresponding template (GPS-TBA or GPS-THA) were fit using a logistic function ( Figure S17). From the fitting equation, the concentration corresponding to the fluorescence response of three blank measurements was used to determine the limit of blank (LOB). Three repeat measurements of the lowest concentration used (0.5 µM for GPS-TBA and S-13 2.49 µM for GPS-THA) were used to determine the limit of detection (LOD). LOB and LOD were calculated as below [3].
, where � is the concentration corresponding to the average response of 3 blank measurements (MIP particles without analyte) and is the standard deviation of these measurements , where is the standard deviation of 3 measurements of the lowest concentration of analyte used.

Uncertainty budget calculations
Because of the multiplicative and quotient forms of the respective equations and because correlations between the quantities are assumed to be negligible, the summation of the squares of the relative uncertainties was performed [4,5]. Because Figure S20 shows the results of biphasic experiments carried out in conventional 10x10 mm cuvettes these fluctuations stem from the mixing process and microscopically non-complete phase separation or small droplets still adhering to the cuvette wall through which the fluorescence is measured (90° geometry of the fluorometer) after the solution was allowed to settle and phase-separate. Because the mixing/phase separation procedure was repeated after each step of addition, fluctuations are significant. However, in order to be able to obtain detailed spectral information at every point of template addition, it was necessary to carry out these experiments with a fluorometer. The experiments have been performed several times, revealing that the major contribution to the error is the repeat uncertainty based on the above-mentioned issues. However, as we have recently shown [6], such problems with 10x10 mm cuvettes can be strongly reduced when working with microfluidic setups. λex = 385 nm.